The present invention relates to an improved method for the purification of alpha-1-acid glycoprotein, and to therapeutic uses of highly purified alpha-1-acid glycoprotein.
Alpha-1-acid glycoprotein (AAG) is a plasma glycoprotein of approximate molecular weight 41 kD. It is an acute phase protein, present in plasma at a concentration of between 0.5-1 g/l in healthy people, rising in disease states, particularly inflammatory diseases, to levels up to about 2 g/l.
The physiological role of AAG is poorly understood. As an acute phase protein, its serum level increases in response to a number of stresses and insults including infection, trauma, burns, etc. AAG is known to act on a wide variety of cells and it has been suggested that AAG may play a role in the immune response. In addition, AAG has been shown to bind to a diversity of drugs, particularly basic and lipophilic drugs. Therapeutic uses of AAG based on this latter aspect have been suggested in the literature but none have been actually developed as far as the clinic.
We believe that one reason for this is the relatively high level of contaminants which remain even in so-called highly purified preparations. The endotoxin lipopolysaccharide (LPS) derived from bacterial cell walls, also known as pyrogen, is one such contaminant.
LPS is the causative agent of septic shock, which is a major cause of morbidity following gram-negative bacterial infection, particularly in hospitalised and immunocompromised patients. The presence of LPS in AAG preparations renders them unsuitable for human therapy.
Currently available methods of purifying AAG are laborious and time consuming, involving a large number of individual steps. Furthermore, they are unsuitable for large scale preparative processes. One such technique is described by Hao and Wickerhauser (Biochem. Biophys. Acta, 322, 99-108 (1973)). This involves adsorption and elution of a Cohn Fraction V supernatant from DEAE Sephadex, concentration, dialysis, adsorption and elution from carboxymethylcellulose, dialysis and finally freeze drying. With both dialysis steps taking 48 hours each, the whole process takes over a week. Furthermore, despite Hao and Wickerhauser""s suggestion to the contrary, the technique is not amenable to scale up for the treatment of the volumes of starting material handled by commercial manufacturers (typically several batches per week of up to 10,000 1 per batch of Cohn Fraction V supernatant). Most importantly this process has not been able to reduce the levels of bound contaminating LPS to levels acceptable for clinical use.
Other prior processes for purifying AAG have not been successful in depleting LPS from AAG preparations to levels which are acceptable for clinical use. One such method involves adsorption and elution of AAG preparations from Detoxigel resins (Boutten et al Eur. J. Immunol. 22, 2687-2695 (1992)). The purpose of this method was to ensure LPS was depleted from an AAG preparation for use in in vitro studies examining the effects of added LPS on cytokine production. This chromatography medium is not however suitable for use in preparing products for human administration, and in any event, LPS levels were only reduced to 200 pg/mg (approx. 2 EU/mg) of protein (EU=endotoxin units). This level is still too high for products intended for human use, particularly at the AAG doses likely to be required clinically (for example from 10 g to 30 g per dose) e.g. in the treatment of drug toxicity.
We have now developed a new process for removing LPS from an AAG containing preparation.
Thus in its broadest aspect, the present invention provides a method of removing LPS from an AAG containing preparation comprising contacting said preparation with a finely divided non-toxic resin.
In this way, it is possible to deplete LPS from AAG containing preparations to levels which are compatible with therapeutic uses of the preparations.
Preferred resins are non-substituted resins.
Preferably, said resin is a particulate resin, especially an inorganic particulate resin and more preferably a hydrophilic resin. Resins with porous surfaces for example silane-based resins such as fumed silica are particularly suitable. One such fumed silica resin which may be used in the method of the invention is the commercially available fumed silica product AEROSIL(trademark) fumed silica (Degussa AG, Frankfurt), which has siloxane and silanol groups on the surface of the particles.
AEROSIL(trademark) fumed silica and similar resins have previously been used in the pharmaceutical industry both as a component, for example in the formulation of tablets and ointments, and also in purification processes such as the removal of lipid and lipid-like substances, and lipoprotein from plasma and plasma derived products. We are not aware of any previous suggestion to use AEROSIL(trademark) fumed silica, or any other finely divided particulate resin as a depyrogenating agent for AAG. The non-toxic nature of AEROSIL(trademark) fumed silica represents a distinct advantage over prior methods of purifying AAG which rely on separation techniques using materials which are not suitable for therapeutic applications.
For use in the process of the invention, the particles may have a high surface to weight ratio such as from 1 m2/g to 1000 m2/g, preferably from 50 m2/g to 700 m2/g, and more preferably from 330 m2/g to 430 m2/g, such as 380 m2/g.
We have also developed a new simple purification method for AAG which includes our new depyrogenation step and which produces a depyrogenated AAG preparation suitable for clinical use. The new purification method accordingly overcomes the aforementioned disadvantages associated with prior AAG purification processes.
Thus in another aspect, the present invention provides a method of purifying AAG comprising contacting an AAG-containing preparation with an anion exchange matrix, eluting an AAG-enriched fraction from said matrix and depyrogenating an AAG-enriched fraction by contact with a finely divided non-toxic particulate resin followed by elution of an LPS-depleted AAG fraction.
Using such a technique, AAG preparations containing as little as 0.016 EU/mg AAG protein can be obtained.
A variety of AAG containing starting materials may be used, for example plasma, cryosupernatant, and plasma fractions for example Cohn Fraction V supernatant and Cohn Fraction IV supernatant. In the case of recombinant AAG production, the technique may also be used on cell cultures and cell culture supernatants and fractions thereof. For reasons of economy, Fraction V supernatant is a preferred starting material, since this enables maximum usage of donated plasma, the fraction essentially being a waste product in the purification of albumin, and being particularly rich in AAG. Fraction V supernatant typically contains 40% ethanol, 10 mM citrate, 50 mM acetate pH 4.8. It has a low protein content ( less than 2 g/l); 80% of the UV absorbing material (OD280) has a molecular weight  less than 10,000 daltons. AAG has a relatively low molecular weight and is extremely soluble; it does not precipitate during the Cold Ethanol Fractionation Process hence the majority (xcx9c60 to 80%) is found in solution in Fraction V supernatant. Typical AAG concentrations in Cohn Fraction V supernatant will be in the range 0.2 to 0.35 g/l.
Any conventional anion exchanger may be used, provided, of course, that it has the ability to bind AAG. Examples include inert substrates such as agarose, for example Sepharose carrying functional groups having the ability to bind AAG such as positively charged groups for example diethylaminoethyl (DEAE), diethyl-(2-hydroxypropyl)-aminoethyl (QAE) and quaternary ammonium (Q). High capacity resins are preferred, and preferably resins of larger particle size, in the range 100 to 300 xcexcm. The increased bed stability of large beads is of advantage in treating viscous materials such as Fraction V supernatant allowing minimal back pressure when the process is carried out by column chromatography; furthermore, AAG-containing fractions can be rapidly removed, maximising AAG recovery and reducing the process time. Preferred matrices include Q-Sepharose Big Bead, Q Hyper D and Toyopearl Super Q. All have high capacities for AAG.
The AAG containing starting material may conveniently be contacted with the anion exchanger in the presence of an ethanolic solution of concentration from 30 to 45% preferably 35 to 45%, more preferably at about 40%, at the pH of fraction V supernatants, about pH 4.5 to 5.5, no adjustment being required, and at temperatures in the range of 2xc2x0 to 30xc2x0 C., preferably 5 to 15xc2x0 C. and more preferably about 10xc2x0 C.
The anion exchange matrix may be provided either as batch or column form, the latter being preferred for both speed and convenience.
Generally, the matrix is used in a ratio of AAG-containing material to matrix from 1000:1 to 5:1 conveniently about 200:1 (by volume).
In operating the method as a column, the anion exchange medium will normally be packed into the column and then equilibrated with a relatively low ionic strength buffer at a pH in the range 4.0 to 5.5, preferably 4.0 to 4.8 and more preferably about 4.1. A useful buffer is acetate buffer, for example sodium acetate of concentration 0.02 M to 0.2 M preferably 0.1 M to 0.13 M and more preferably about 0.13 M.
After loading the AAG containing material onto the column non-bound proteins may be removed by washing with a low ionic strength buffer for example the buffer used to pre-equilibrate the column.
AAG may then be eluted in a variety of ways. One such way is by means of increasing ionic strength buffers based on the equilibration buffer. Generally, the electrolyte is sodium chloride, but other salts may be used for example sodium acetate. AAG may be eluted either by means of a linear salt gradient or by a stepwise increase in salt concentration, from 0 to saturated ( greater than 3M) sodium chloride, preferably 0 to 1.0 M and more preferably gradients of 0-0.2 M. A useful buffer for eluting AAG is 0.13 M sodium acetate 0.2 M sodium chloride pH 4.1.
In an alternative method, AAG may be eluted by decreasing the pH of the buffer to below pH 4.1, for example by adding appropriate buffers such as 0.1 M sodium phosphate, at a pH in the range 2.0-3.0.
The AAG enriched preparation is then neutralised with sodium hydroxide prior to depyrogenation according to the invention and as described below.
Depyrogenation with finely divided particles as described previously may be carried out conveniently as a batch process, using equipment which has been depyrogenated according to conventional methods for example soaking in alkali such as 0.5 M NaOH for at least one hour or by heating at temperatures above 200xc2x0 C. for greater than one hour.
Generally, the partially purified AAG preparation will be contacted with the particles for upwards of 15 minutes to an overnight contact time, e.g. for several hours e.g. 2 hours and generally for about 1 hour, at temperatures of between 4xc2x0 to 70xc2x0 C., preferably 40 to 37xc2x0 C. and more preferably about 20xc2x0 C.
The particles may be used in a weight:weight ratio of particles to AAG protein of from 50:1 to 0.2:1 (by weight), preferably from 5:1 to 0.1:1 preferably 2:1 to 1:1 and more preferably 1:1 with AAG being in solution at a concentration of 0.1 g/l to 250 g/l preferably 2 to 50 g/l and more preferably about 3 g/l.
The AAG preparation may be concentrated for therapeutic use using conventional methods including ultrafiltration or freeze drying. Ultrafiltration (UF) may conveniently be carried out using a 10 kD UF cassette (ie. a filter which has a 10,000 dalton molecular weight cut off) using tangential flow, to reach a concentration in the range 10 to 250 g/l, conveniently 100 g/l.
Depyrogenation may take place either before or after this concentration step, however we have found that concentration after depyrogenation is preferred as it reduces losses of AAG at depyrogenation and improves yields.
To further process the purified AAG into a form suitable for therapeutic use, the preparation may be diafiltered into an appropriate buffer suitable for human administration, for example phosphate buffered saline at pH 7.5.
The AAG preparation may be subjected to a range of viral inactivation steps, which are now a mandatory requirement in most countries for blood and plasma derived products. We have found that AAG purified according to the process of the invention is stable to prolonged heating at high temperatures. Thus a preferred viral inactivation step comprises heating the purified AAG in solution at pasteurisation temperatures of from 58xc2x0 C. to 70xc2x0 C. for at least 2 hours, preferably for about 10 hours, optionally in the presence of recognized stabilisers such as salts, amino acids or sugars examples of which include sodium chloride, glycine and sucrose, although stabilisers are not absolutely required for such AAG preparations.
Compared to the majority of proteins, AAG is a relatively small molecule; thus another method of eliminating viruses from an AAG containing preparation according to the invention is virus filtration, through filters of pore size  less than 50 nm, preferably 15 nm.
Current recommendations from the Committee for proprietary medicinal products European Commission are for two independent virus inactivation/elimination steps for intravenous products made from plasma. Both of these methods can be used sequentially for treating the AAG preparations according to the invention. Other methods which can be used include solvent detergent treatment e.g. as described in Edwards et al., Vox. Sang. 52, 53-59 (1987) and heat treatment of freeze-dried AAG preparations according to the invention.
By using the process of the invention, we have been able to purify AAG from Fraction V supernatant with an average yield of up to 80% and purity of  greater than 98% as measured by cellulose acetate electrophoresis. Furthermore, using this method it is possible to produce, for the first time, substantially depyrogenated AAG preparations having a LPS concentration of less than 0.1 Eu/mg protein, which passes the European Pharmacopoeia animal pyrogenicity test for substances of this kind.
Thus viewed from a further aspect, the present invention provides AAG substantially free of LPS, said AAG having a LPS concentration of less than or equal to 0.1 Eu/mg AAG, preferably less than 0.075 Eu/mg and more preferably less than 0.050 Eu/mg and especially preferably less than 0.02 Eu/mg. Such an AAG preparation substantially depleted of LPS according to the invention is hereinafter referred to as Apo-AAG.
According to a further aspect, the present invention provides a virus inactivated or depleted Apo-AAG preparation. Inactivation may be carried out by methods including the aforementioned solvent detergent treatment, or pasteurisation, and depletion methods include the aforementioned virus filtration.
According to a yet further aspect, the present invention provides Apo-AAG according to the invention for use in therapy.
AAG is known to have useful drug binding properties, and our new highly purified Apo-AAG is particularly useful in the clinical management of drug overdoses, for example in the case of tricyclic anti-depressants where overdose can be lethal.
Thus viewed from a further aspect, the present invention provides a method of treating drug toxicity comprising administering to a patient in need of such treatment an effective amount of Apo-AAG.
In another aspect, the present invention provides the use of Apo-AAG in the manufacture of a medicament for use in the treatment of drug toxicity.
This aspect is particularly useful in treating toxic effects associated with overdoses of basic drugs such as quinine, lignocaine, propranolol and particularly tricyclic anti-depressants such as amitriptyline, desipramine and nortriptyline.
For therapy according to the invention, AAG may be formulated according to conventional methods of pharmacy, together with pharmaceutically acceptable excipients, carriers or diluents as, for example, described in Remingtons Pharmaceutical Sciences ed Gennaro, Mack Publishing Company, Pennsylvania USA (1990). Additional components such as preservatives may be used. AAG may be formulated into compositions for administration by any convenient route e.g. enterally or parenterally, by transmucosal delivery e.g. rectally, in implants or by intravenous, intramuscular or subcutaneous injection etc.
Viewed from a further aspect, the present invention provides a pharmaceutical composition comprising AAG together with one or more pharmaceutically acceptable carriers or excipients.
These compositions may for example take the form of solutions, emulsions, pessaries and suppositories, as well as other stabilised presentations such as freeze dried plugs, foams and glasses. The formulation may be chosen as appropriate to the route of administration which may be by all conventional methods including parenterally (e.g. intrperitoneally, subcuutaneously, intramuscularly, intradermally or intravenously or mucosally (e.g. orally, nasally, vaginally, rectally and via the intraocular route).
Actual treatment regimes or prophylactic regimes and dosages will depend to a large extent upon the individual patient and may be devised by the medical practitioner based on individual circumstances. Doses may be in the range of 10 to 30 g AAG.